1. Field of the Invention
The present invention relates to methods for generating transgenic animals, particularly transgenic birds and fish, using viral constructs engineered to carry the transgene(s) of interest.
2. Description of the Related Art
Early transgenic experiments used an oncoretrovirus to introduce the gene of interest into embryonic cells (Jaenisch Proc. Natl. Acad. Sci. USA 73:1260-1264 (1976)). In a typical experiment an engineered Moloney strain of mouse leukemia virus (MoMLV) was injected into the blastocyst cavity of mice. While the transgene was often integrated into the genome of the resulting mice, no gene expression could be detected.
Today, the majority of transgenic animals are made using direct injection technology (Gordon and Ruddle Science 214:1244-1246 (1981)). Briefly, a DNA construct carrying the gene of interest is injected directly into the pronucleus of a single-cell zygote. The cell is then implanted into a pseudo-pregnant female and the resulting progeny is analyzed for expression of the gene.
While this method achieves both integration and expression of the transgene, there are a number of significant drawbacks to the direct injection technique. First, in order to carry out the technique it is necessary to inject DNA directly into the pronucleus. This is possible in some specific strains of mice, such as Black6×BDA, because the male pronucleus is visible. However, in other animals and other strains of mice the pronucleus is less visible, making the technique extremely difficult. Further, the injection requires the assistance of a skilled technician and a significant investment in equipment; micromanipulators are necessary to hold the cell and the injection pipette and a pressure source is required that can deliver picoliter amounts of DNA solution.
A second equally significant problem with the direct injection method is the low percentage of injected zygotes that produce transgenic animals. The injection pipette must go through the zona pellucida, the cell membrane and the nuclear envelope. Thus only 80-90% of mouse cells survive the injection. Other animal cells are less hardy and the survival rates are somewhat lower, with about 60% survival for rats and 40-50% for cows. In mice, of the original zygotes, about 25% are successfully injected and implanted in a pseudopregnant female. About 20% of the resulting animals have the transgene integrated into their genome. Of these, about 20% will express the gene. However, even if the animals express the gene, it is possible that the expression pattern will not be useful. Thus, only about 1% of injected zygotes result in transgenic animals that express the gene of interest. This low efficiency of transgenesis is particularly troubling for larger animals, such as pigs, cows or goats, in which obtaining large numbers of embryos is not possible (see, e.g., Wall et al. J. Cell. Biochem. 49:113 (1992)).
In addition, direct pronuclear injection is not possible for many types of animals, including birds.